ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1994, p. 2838-2842 Vol. 38, No. 120066-4804/94/$04.00+0Copyright X 1994, American Society for Microbiology

Extended-Spectrum 3-Lactamases in Clinical Isolates ofEnterobacter gergoviae and Escherichia coli in China

YULIN CHENG AND MINJUN CHEN*Department of Clinical Laboratories, Peking Union Medical College Hospital, 1 Shuaifuyuan,

Beijing 100730, Peoples Republic of China

Received 1 November 1993/Returned for modification 2 February 1994/Accepted 26 September 1994

Resistance to ceftazidime, detected in isolates of Escherichia coli 5518 and Enterobacter gergoviae 3773 fromour hospital, was transferred, together with resistance to aminoglycosides, trimethoprim, sulfonamide, andother P-lactam antibiotics, by conjugation to E. coli JP559. Both E. coli transconjugants were resistant toampicillin, all cephalosporins, and aztreonam but remained susceptible to cefoxitin and imipenem. Theenzymes of the two transconjugant strains readily hydrolyzed cephalosporins in a spectrophotometric assay.Hybridization results suggested that the extended-spectrum I-lactamase produced by E. coli 5518 was anon-TEM, non-SHV enzyme, the origin of which is currently unknown. The j-lactamase produced by E.gergoviae 3773 was of the SHV type and was further proved to be SHV-2 by DNA sequencing. Thus,extended-spectrum I-lactamases are occurring in China as well as in other parts of the world.

The extended-spectrum cephalosporins and other new 1-lac-tams, such as monobactams, were once thought to be poorlyhydrolyzable by most 3-lactamases. Thus, they have been usedextensively clinically. In some species, such as Pseudomonasaeruginosa and Enterobacter cloacae, emergence of resistanceduring therapy with one of these new 3-lactams by overpro-duction of chromosomally mediated ,-lactamase was docu-mented (17, 25, 26). The first extended-spectrum 1-lactamase,designated SHV-2, was reported in Germany in 1983 (3, 14).Since then, more and more extended-spectrum 1-lactamaseshave been detected across the world (11, 15, 22). In this study,we describe two extended-spectrum ,-lactamases in multire-sistant isolates of Enterobacter gergoviae 3773 and Escherichiacoli 5518 from Peking Union Medical College Hospital.

MATERIALS AND METHODS

Bacterial strains and plasmids. Strains and plasmids used inthis study are listed in Table 1. E. gergoviae 3773 and E. coli5518 (harboring resistant plasmids pC3773 and pC5518, re-spectively) were isolated from abdominal abscesses in twopostsurgery patients at Peking Union Medical College Hospi-tal in 1991. E. coli HB101 was used for cloning. E. coli JM101was utilized as the recipient for M13mpl9 recombinant bacte-riophage for nucleotide sequencing. Strains were identified bythe API 20E system (BioMerieux, Marcy l'Etoile, France).

Antibiotics. Antibiotics used were rifampin, cephaloridine,and cefazolin (Tuobin Pharmaceutical Works); nalidixic acid(Boshan Pharmaceutical Works); ampicillin (Shanghai no. 4Pharmaceutical Works); cefuroxime and ceftazidime (GlaxoPharmaceuticals, Ltd.); cephradine (E. R. Squibb & Sons Inc.);cefoperazone (Pfizer Inc.); ceftriaxone (F. Hoffmnann-La RocheInc.); cefotaxime (Changzheng Pharmaceutical Works); cefox-itin (Merck Sharp & Dohme); and clavulanate (BeechamLaboratories).

Susceptibility testing. MICs of 3-lactam antibiotics weredetermined by the agar dilution method in Mueller-Hintonagar with an inoculum of 105 to 106 CFU, alone or in

* Corresponding author. Mailing address: Department of ClinicalLaboratories, Peking Union Medical College Hospital, 1 Shuaifuyuan,Beijing 100730, Peoples Republic of China. Fax: 0086-1-512-4875.

combination with clavulanate (2 jxg/ml). Plates were incubatedat 37°C for 18 h, and the MIC was recorded as the lowestconcentration that totally inhibited growth, disregarding asingle colony or a faint haze caused by the inoculum (19). Agardisk diffusion susceptibility tests were performed according tothe guidelines of the National Committee for Clinical Labora-tory Standards (20).

Conjugation. Conjugation experiments were carried out bythe membrane filter mating technique (5). The recipient was E.coli K-12 JP559 (Rif Nalr). A 0.5-ml volume of the donorculture and 1.0 ml of the recipient culture were mixed in amicrocentrifuge tube, and the tube was centrifuged at 12,352 xg for 3 min. The cell pellet was resuspended in 50 [LI of brainheart infusion (BHI) broth, transferred onto a 2.5-cm-diame-ter, 0.45-,um-pore-size filter on a prewarmed BHI agar plate,and incubated at 37°C for 4 to 6 h. The filter was thenimmersed in 2 ml of BHI broth, the cells were resuspended byscraping with a wire loop, and 0.2 ml of the resuspension wasspread on transconjugant-selecting plates containing ceftazi-dime (12 ,ug/ml), rifampin (380 ,ug/ml), and nalidixic acid (380LgIml).13-Lactamase assay. ,B-Lactamases were obtained from

transconjugants as follows (23, 28, 31). Overnight cultures inBHI broth were diluted 100-fold with fresh broth containingampicillin (20 ,ug/ml) or ceftazidime (12 jig/ml) and incubatedwith shaking for 20 h at 37°C. Cells were harvested bycentrifugation at 4°C, washed with 0.1 M phosphate buffer (pH7.0), and resuspended in 1/50 of the original volume. Aftersonication at 0°C, crude extracts were obtained by ultracentrif-ugation at 45,000 x g at 4°C.The 13-lactamase assay was performed spectrophotometri-

cally by measuring the change in absorbance at the appropriatewavelength for each substrate. The wavelengths were 255 nmfor cephaloridine; 265 nm for cefazolin and cefuroxime; 275nm for cefoperazone; 257 nm for ceftriaxone, cefotaxime, andceftazidime; and 260 nm for cefoxitin. ,B-Lactamase activitywas determined in 3 ml of a 100 ,uM substrate in 10 mMphosphate buffer, pH 7.0. Substrate blanks were recorded foreach reaction.One unit of activity was defined as the amount of enzyme

that hydrolyzed 1 ,uM cephaloridine per min at 37°C and pH

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TABLE 1. Bacterial strains and plasmids used in this study

Strain Plasmid Relevant markerse Source

E. coli 5518 pC5518 CaZr Kanr Strr Genr Tmpr Clinical isolateE. gergoviae 3773 pC3773 CaZr Kanr Strr Tmpr Clinical isolateE. coli JP559 Rif Nalr J. PittartE. coli C5518 pC5518 Cazr Kanr Strr Genr Tmpr Rif Nalr This workE. coli C3773 pC3773 CaZr Kanr Strr Genr Tmpr Rif Nalr This workE. coli HB101 pMON38 Ampr (SHV-1) Cm G. A. JacobyE. coli C600 pBR322 Ampr (TEM-1) Tetr G. A. JacobyE. coli HB101 pCHEN1 Tetr Cazr This workE. coli HB101 C. F. HuangE. coli JM101 C. F. HuangE. coli ATCC 25922 American Type Culture

Collection

a Abbreviations: CaZr, resistance to ceftazidime; Tmpr, resistance to trimethoprim-sulfamethoxazole; Kanr, resistance to kanamycin; Strr, resistance to streptomycin;Genr, resistance to gentamicin; Rifr, resistance to rifampin; Nalr, resistance to nalidixic acid; Ampr, resistance to ampicillin; Cm, resistance to chloramphenicol; Tetr,resistance to tetracycline.

7.0. The rates of hydrolysis calculated were relative to that ofcephaloridine (100%).DNA agarose gel electrophoresis. Plasmid DNA was ex-

tracted from clinical isolates and transconjugants by the rapidprocedure of Takahashi and Nagano (29), and the crudeextract was used directly for electrophoresis, which was per-formed in a 0.7% agarose gel for 2 h at 10 V/cm. The size of theplasmids was roughly determined by using pMON21 (7.5 kb).

Nucleic acid techniques. For large-scale preparations, plas-mid DNA was extracted from a 1-liter culture and purified byultracentrifugation in cesium chloride-ethidium bromide dis-continuous gradients (24). Plasmid DNA was digested withrestriction endonuclease according to the recommendations ofthe manufacturers. The 420-bp BglI-HincII fragment and the360-bp PvuII-PvuII fragment (internal to the structural genes

for TEM-1 of pBR322 and for SHV-1 of pMON38, respec-tively) and the 450-bp PvuII-ClaI fragment (containing the

partial structural gene for SHV-1 of pMON38) were used as

probes (10, 18). DNA was labeled with [ot-32P]ATP by nicktranslation, and hybridization was carried out under stringentconditions after DNA was transferred to nitrocellulose sheetsby the method of Southern (10). Cloning and subcloning werecarried out as described by Sambrook et al. (24). DNAsequencing was performed by the dideoxy polymerase chaintermination method of Sanger et al. (8, 27).

RESULTS

Antibiotic susceptibilities. The resistance phenotypes of theclinical isolates of E. gergoviae 3773 and E. coli 5518 were

determined by the disk diffusion assay (results not shown).These two isolates were highly resistant to ampicillin, piperacil-lin, carbenicillin, cephalothin, cephaloridine, cefazolin, andcefuroxime. E. gergoviae 3773 was moderately susceptible to

TABLE 2. MICs of P-lactams for E. coli and its derivatives producing different plasmid-mediated 3-lactamasesMIC (,ug/ml) for strain:

I3Lactama ATCJP559(pC3773) JP559(pC5518) HB101(pMON38) JP559 HB101 259A2

Ampicillin >2,048 >2,048 2,048 4 4 4Ampicillin plus clavulanate 16 32 4 2 2 2

Cefazolin 256 512 8 2 2 1Cefazolin plus clavulanate 1 1 0.5 0.5 0.5 0.5Cephradine 256 256 16 8 8 8Cephradine plus clavulanate 16 8 8 8 8 8

Cefuroxime 128 256 4 4 1 4Cefuroxime plus clavulanate 8 4 2 2 1 2

Ceftazidime 16 128 1 0.125 0.06 0.06Ceftazidime plus clavulanate 1 1 0.125 <0.06 <0.06 <0.06Ceftriaxone 32 >64 <0.06 <0.06 <0.06 <0.06Ceftriaxone plus clavulanate <0.06 <0.06 <0.06 <0.06 <0.06 <0.06Cefotaxime 32 >64 <0.06 <0.06 <0.06 <0.06Cefotaxime plus clavulanate <0.06 <0.06 <0.06 <0.06 <0.06 <0.06Cefoperazone 64 64 4 0.125 0.125 0.25Cefoperazone plus clavulanate 0.5 0.25 <0.06 <0.06 <0.06 <0.06Cefoxitin 1 2 2 2 2 2Aztreonam 8 32 bImipenem <0.5 <0.5

a The concentration of clavulanate is a constant 2 pg/ml.b, not done.

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ANTIMICROB. AGENTS CHEMOTHER.

TABLE 3. Hydrolysis of ,B-lactams by crude extract 1-lactamases

Enzyme Rate of hydrolysis relative to that of CLR (%)'Source of enzymes activity

(U/ml) CLR CZL CRX CFP CTR CTX CAZ FOX

E. coli pc5518 6.1 100 93 72 87 77 75 66 5E. coli pc3773 1.4 100 89 69 82 75 67 61 3E. coli HB101(pMON38) 6.9 100 70 12 65 8 4 6 6

a Abbreviations: CLR, cephaloridine; CZL, cefazolin; CRX, cefuroxime; CFP, cefoperazone; CTR, ceftriaxone; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin.

ceftazidime and ceftriaxone (inhibition zone diameters of 17and 14 mm, respectively) and resistant to chloramphenicol,and E. coli 5518 was resistant to ceftazidime and ceftriaxone.Both of them were also resistant to streptomycin, kanamycin,gentamicin, and trimethoprim-sulfamethoxazole.MICs and substrate profile. MICs of 1-lactams, alone and

combined with clavulanate (2 ,ug/ml), against E. coli transcon-jugants harboring pC3773 or pC5518 compared with thoseagainst E. coli harboring pMON38 (SHV-1) are shown inTable 2. MICs of the extended-spectrum cephalosporins for E.coli harboring pC5518 were slightly higher than those for E.coli harboring pC3773. E. coli transconjugants harboringpC5518 or pC3773 also showed decreased susceptibility toaztreonam (MIC, 16 to 32 ,ug/ml) but remained susceptible tocefoxitin (MIC, 1 to 2 ,ug/ml) and imipenem (MIC < 0.5jig/ml). Activities of ampicillin, cefazolin, cephradine, cefu-roxime, ceftazidime, ceftriaxone, cefotaxime, and cefopera-zone were enhanced by the addition of 2 ,ug of clavulanate perml. The substrate profile of P-lactamase obtained from E. colitransconjugants harboring pC3773 or pC5518 was comparedwith that of SHV-1 and showed an extended-spectrum profile(Table 3). The rates of hydrolysis of agents tested were veryhigh (>61%), with the exception of that of cefoxitin (3 to 5%).

Characterization of the specifying plasmid. Plasmid DNAfrom the clinical isolates of E. gergoviae 3773 and E. coli 5518and their E. coli transconjugants was analyzed by agarose gelelectrophoresis (Fig. 1). A plasmid of 60 kb was isolated fromE. gergoviae 3773 and its transconjugant. E. coli 5518 harboredtwo plasmids, and only the smaller one (pC5518) of <10 kb,

a b c d e f

responsible for the extended-spectrum activity, was transfer-able.

In the E. coli transconjugant harboring pC3773, 3-lactamresistance was cotransferred with resistance to aminoglyco-sides (kanamycin and streptomycin), trimethoprim-sulfame-thoxazole, and chloramphenicol. In the E. coli transconjugantharboring pC5518, 3-lactam resistance was associated withresistance to aminoglycosides (kanamycin, streptomycin, andgentamicin) and trimethoprim-sulfamethoxazole.Under stringent conditions, an intragenic SHV-1-derived

probe hybridized with the largest (19 kb) fragment of theBamHI digest of pC3773; however, no fragment of the EcoRIdigest of pC5518 hybridized with either the intragenic SHV-1-der'ived probe or the intragenic TEM-1-derived probe (Fig. 2).

Cloning and nucleotide sequencing of SHV P-lactamasegene from pC3773. For further studies with the SHV-typeextended-spectrum ,-lactamase of pC3773, we cloned EcoRIfragments of pC3773 into the EcoRI site of pBR322. Thecloned DNA was used to transform E. coli HB101 withselection for ceftazidime. From one of the transformants, weisolated a plasmid, designated pCHEN1, that contained the4.4-kb EcoRI fragment of the vector and a 6.5-kb EcoRIfragment from pC3773 including the extended-spectrum 1-lac-tamase gene.There are SHV-type enzymes that include SHV-1 through

SHV-6, but there are four molecular sequences described forthe SHV-2 enzyme. They differ from one another by pointmutations at amino acid positions 201, 234, and 235, whichwere located in the 0.36-kb PvuII-PvuII fragment and the0.45-kb PvuII-ClaI fragment of pMON38 (18). By using thesetwo fragments of pMON38 as probes, hybridization was per-

a b c d e

is

a b c d e

--or

-7. 6kb

A B C'

a b c d e

FIG. 1. Agarose gel electrophoresis of crude lysates of donors,

recipient, and transconjugants. Lanes: a and b, E. coli transconjugantand E. gergoviae 3773, respectively; c, recipient, E. coli JP559; d and e,

E. coli transconjugant and E. coli C5518, respectively; f, marker,pMON21. Chr, chromosomal DNA.

FIG. 2. Southern blot analysis of R-plasmid encoding extended-spectrum P-lactamases. (A) Autoradiogram of gel in panel B probedwith TEM-1. (B) Gel stained with ethidium bromide. (C) Autoradio-gram of gel in shown in panel B probed with SHV-1. Lanes: a,pMON38 (SHV-1); b, pBR322 (TEM-1); c, pC5518 plus EcoRIfragment; d, pC3773 plus BamHI fragment; e, X DNA plus HindIIIfragment.

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V-LACTAMASES IN E. GERGOVL4E AND E. COLI 2841

TABLE 4. Differences at molecular level between SHV-1 and different SHV-2s

Strain and/or P-lactamase (country)Codon (amino acid) for amino acid position: Source or

31 134 183 234 262 reference

SHV-1 CTA (Leu) CTA (Leu) GCC (Ala) GGC (Gly) ACC (Thr) 3SHV-2K pneumoniae 5214773 GCG (Ala) AGC (Ser) 16

(United States)K ozaenae 2180 (Germany) AGC (Ser) 9S. typhimurium 122 (France) CAA (Gln) AGC (Ser) 4E. gergoviae 3773 (China) CTG (Leu) AGC (Ser) ACG (Thr) This work

formed under high-stringency conditions and two homologousfragments, a 0.3-kb PvuII-PvuII fragment and a 0.5-kb PvuII-ClaI fragment, were observed on pCHEN1. These two homol-ogous fragments of pCHEN1 were subcloned into thepolylinker of the replicative forms of bacteriophage M13derivative mpl9. The recombinant phages were used to trans-fect E. coli JM101, and nucleotide sequences were determined.The open reading frame for the SHV-type enzyme gene was

858 bp long, encoding a protein of 286 amino acids, of whichthe first 21 amino acids were a signal peptide and did notappear in the mature enzyme. The partial nucleotide sequenceof the SHV-type enzyme gene we report here covered all of thestructural gene after codon 86, including the point mutationsresponsible for the extended-spectrum activity. Comparison ofthe deduced amino acid sequence of the enzyme from E.gergoviae 3773 with that of SHV-1 showed only one substitu-tion of serine for glycine at position 234 (Table 4), suggestingthat the extended-spectrum P-lactamase harbored by E. ger-goviae 3773 was SHV-2. There are another two point mutationsat position 134 (CTA to CAG) and position 262 (ACC toACG) which did not change the amino acids (Table 4).

DISCUSSION

According to their molecular structure, plasmid-mediatedcephalosporin-hydrolyzing extended-spectrum ,B-lactamasesdetected in members of the family Enterobacteriaceae may beclassified into several groups: the SHV type derived fromSHV-1, the TEM type derived from TEM-1 or TEM-2, and theAmpC type derived from the chromosomal AmpC 3-lacta-mases such as CMY-2 (11). The activities of SHV-type andTEM-type extended-spectrum ,-lactamases can be inhibitedby the classical P-lactamase inactivators clavulanic acid andsulbactam, and these enzymes do not hydrolyze cephamycinsand imipenem well. The AmpC type is resistant to the classical,B-lactamase inactivators and cephamycins. Recently, two novelplasmid-mediated extended-spectrum r-lactamases were re-ported to be present in P. aeruginosa (6, 21). The first novel,-lactamase is a carbapenem-hydrolyzing metalloenzyme resis-tant to inhibition by clavulanate and belonging to the class B3-lactamases which confers resistance to imipenem as well as

to ceftazidime and moxalactam (21). The second recentlyreported P-lactamase is a non-TEM, non-SHV enzyme calledOXA- 1 which is a derivative of a plasmid-mediated oxacillin-hydrolyzing enzyme, OXA-10 (PSE-2), belonging to the classD P-lactamases and whose activity is not inhibited by clavu-lanate (6). DNA-DNA hybridization shows that the cephalo-sporin-hydrolyzing extended-spectrum 3-lactamase harboredby E. coli 5518 is a non-SHV-, non-TEM-type enzyme, and it isunlikely that it is derived from the other P-lactamases men-tioned above because of its sensitivity to clavulanate andcefoxitin. This enzyme produced by E. coli 5518 has hydrolytic

properties that resemble those of the extended-spectrum B-lac-tamase harbored by E. gergoviae 3773 (Tables 3 and 4), and onefragment of pC5518 hybridizes both with the intragenic SHV-1-derived probe and with the intragenic TEM-1-derived probeunder low-stringency conditions (results not shown), suggest-ing that it may be classified within the class A J-lactamases.This needs to be studied further. Recently, a non-SHV,non-TEM plasmid-mediated extended-spectrum ,-lactamase,MEN-1, was also reported to be present in E. coli, and proteinsequence analysis of MEN-1 revealed that it shares 72%identity with the chromosomally mediated 1-lactamases ofKiebsiella oxytoca (2). In addition, pC5518 is less than 10 kblong, too small to be a self-transferable plasmid, and it seemslikely that it was mobilized by the larger plasmid in the samestrain (30).A resistant isolate of Klebsiella pneumoniae from Hua-Shan

Hospital in Shanghai, China, which produced ,-lactamase withthe isoelectric point and inhibition properties of SHV-2, hasbeen reported (12). SHV-2 has been detected in Kiebsiellaozaenae and S. typhimurium (Table 4), and all the enzymesshare the same Gly--Ser change at position 234 in comparisonwith SHV-1 (4, 9, 11, 16). The nucleotide sequences of SHV-2variants from different sources have a few unique pointmutations at other positions. One of these nucleotide muta-tions causes an amino acid substitution but does not affect theenzyme activity, and the others do not even affect the aminoacids encoded. SHV-2 harbored by E. gergoviae 3773 possessesat least two more point mutations at positions 134 and 262besides the critical mutation at position 234, but these muta-tions (CTA to CTG for position 134 and ACC to ACG forposition 262) do not cause amino acid changes. On the basis ofgenetic similarity (9), SHV-type enzyme may have evolvedfrom chromosomal ,-lactamase LEN-1 ofK pneumoniae, andthe two enzymes both belong to class A in the scheme ofAmbler. Arakawa et al. demonstrated a close evolutionaryrelationship between TEM ,B-lactamases and LEN-1 (1). Ac-cording to the recently published three-dimensional structureof the class A ,B-lactamase TEM-1 from E. coli (7, 13), position234 is located near the substrate-binding site. For SHV-2, thehydroxyl group of Ser-234 may interact directly with the sidechain of the new 3-lactams via a hydrogen bond, whichtherefore increases catalytic activity toward them.

ACKNOWLEDGMENTS

This work was supported in part by the National Natural ScienceFoundation of China.We thank G. A. Jacoby for bacterial strains, plasmids, and help. We

are very grateful to Boqin Qiang for providing restriction endonucle-ases and technical advice. We are also grateful to the ClinicalMicrobiology Division, Peking Union Medical College Hospital, fortechnical assistance.

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